The aim of this proposal is to contribute to an understanding of the processes involved in regulation of iron homeostasis. Iron is required by most organisms for survival and plays a role in many biological processes including oxygen and electron transport, nitrogen fixation, and DNA synthesis. Free iron is toxic to cells due to its ability to form reactive hydroxyls that cause peroxidation of lipid membranes and damage DNA. In human, the accumulation of excess cellular iron can result in tissue damage leading to cirrhosis, arthritis, cardiomyopathy, diabetes mellitus and increased risk of cancer. To prevent iron toxicity, cells tightly maintain iron levels by regulating the amount of iron taken up and stored by cells. Cellular iron homeostasis is regulated by the iron- regulatory proteins 1 and 2 (IRP1 and IRP2). IRPs are cytosolic RNA- binding proteins that bind to a specific stem-loop structure termed the iron responsive element (IRE). IREs are located in the 5' or 3' untranslated regions of mRNAs that encode proteins involved in uptake, storage or utilization of iron. IRP1 exhibits two mutually exclusive activities depending on intracellular iron levels: when iron is scarce IRP1 binds to IREs regulating mRNA translation or stability; when iron is abundant IRP1 exhibits aconitase activity catalyzing the interconversion of citrate and isocitrate. In contrast to IRP1, IRP2 functions solely as an RNA-binding protein. Although IRP1 and IRP2 both regulate IRE-containing mRNAs these proteins differ biochemically and structurally and iron regulates their activities by different mechanisms. It is not known why two IRPs evolved not is it know whether they play different roles in regulating iron metabolism. In this proposal, we plan to address the different roles of IRP1 and IRP2 in iron metabolism.